Urinary tract infections (UTIs) result in considerable morbidity and health care costs. The most common cause is the Gram-negative bacteria, uropathogenic Escherichia coli (UPEC). Human infections are characterized by bacteriuria and the release of cytokines, exfoliated cells, and polymorphonuclear leukocytes (PMN) into the urine. These clinical manifestations of disease are all seen in mice. Using qRT-PCR, microarray analyses, microscopy, and immunohistochemistry, we have further described the response to UPEC infection. We have demonstrated that type 1 pili-mediated UPEC binding to and invasion of bladder epithelial cells potentiates toll-like receptor (TLR) 4-mediated signaling and results in rapid upregulation of genes involved in cell differentiation, proliferative and immediate-early responses, pro-inflammatory responses, apoptosis, stress responses, signal transduction, cell-cell contacts, and metabolic changes. Further, we have shown that TLR4-mediated signaling on both stromal and hematopoietic cells is required for efficient clearance of bacteria. Hematopoietic cells include macrophages (M), dendritic cells (DC), granulocytes, and B and T cells. In this proposal we describe experiments to build on our previous work to understand host processes important in determining the outcome of an encounter between pathogens and the normally sterile host urinary tract. We will delineate the host response to UPEC, including the kinetics of cytokine production within the infected bladder, the hierarchy of infiltration of immune cells, and real time visualization of early interactions between immune cells and infecting bacteria. We will also broaden our understanding of UTIs and host response by characterizing the host response to two Gram-positive bacteria that also cause UTIs in humans, Staphylococcus saprophyticus and Enterococcus faecalis. Differing host response mechanisms and bacterial tropisms between Gram-negative and Gram- positive UTIs may have implications for disease symptoms, progression, and treatment. Using mouse mutant backgrounds and cell and cytokine depletion experiments, we will examine the roles of host immune cells and defense molecules in inflammation, bacterial clearance, reservoir maintenance, and epithelial exfoliation and regeneration. A detailed understanding of the host response to UTIs may lead to elucidation of new and better ways to evaluate, treat, and prevent these common infections. PUBLIC HEALTH RELEVANCE: UTIs occur frequently in otherwise healthy women and result in an estimated ~$2.5 billion annually in health care costs. In this proposal we describe experiments to broaden our understanding of UTIs by characterizing the soluble mediators and host immune cells involved in the host response to the most common cause of UTI (uropathogenic Escherichia coli) and two Gram-positive bacteria (Staphylococcus saprophyticus and Enterococcus faecalis). A better understanding of the host factors involved in determining the outcome of infection is needed to better diagnosis, treat and prevent this common disease.